Single-cell RNA-seq provides insight into the underdeveloped immune system of germ-free mice

Background Germ-free mice feature a profoundly underdeveloped immune system. Despite recent studies that emphasize the role of specific bacteria-derived metabolites in immune cell development and differentiation, it remains unclear how the lack of microbiota leads to immune deficiencies. Results Here we performed droplet-based single-cell RNA sequencing to analyze the bone marrow and peripheral blood of both germ-free and specific-pathogen-free mice, identifying 25 distinct cell types. Our findings indicate that neutrophil apoptosis in germ-free mice is strongly associated with the absence of niacin dehydrogenase, which derived primarily from Pseudomonas . In addition, germ-free mice exhibited elevated excretion of 5’-methylthioadenosine, increased ERK activation induced by reactive oxygen species, and cessation of the bone marrow stromal antigen 2 signaling pathway in germ-free mice. The responses of monocytes and CD8 + T cells to interferon β and interferon γ were reduced in germ-free mice, which accounted for their increased susceptibility to viruses. Conclusions Together, we identified a regulatory mechanism connecting immunodeficiency to the absence of microbiota in germ-free mice and validated these findings via multiple techniques.